The present invention relates to methods and assays for antioxidant detection and, more specifically, to methods and assays that reliably measure the antioxidant capacity of dietary products.
In recent years, much attention has been drawn to antioxidants because of their supposed ability to fight cancer, promote health and prevent a wide variety of diseases including heart disease, aging, and neurodegenerative diseases such as Parkinson's and Alzheimer's. Scientific knowledge and epidemiological evidence suggest that consumption of antioxidant compounds reduces oxidative stress created by reactive oxygen and nitrogen species (“ROS” and “RNS”), and thus provides prophylactic and therapeutic benefits in the treatment of disease. However, the true antioxidant capacity of any given compound and dietary food product is questionable. A current limitation is the lack of validated assays that can reliably and accurately measure antioxidant capacity. Conventional antioxidant assays involve the use of radical generation systems, colorimetric dyes, fluorophores or enzymes, and require spectroscopic or electrochemical instrumentation. In general, these assays are not portable and their cost is prohibitive for general use. Additionally, all of these assays determine antioxidant capacity in a controlled laboratory environment.
Most antioxidant activity assays currently in practice assess the ability of an antioxidant to scavenge synthetically created free radicals (e.g. ROS, RNS) or to reduce reactive redox metals (e.g. iron, copper or gold). Some assays assess the ability of an antioxidant to scavenge a specific radical (e.g. ABTS.+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), DPPH. (1,1-diphenyl-2-picrylhydrazyl), superoxide (O2.−), while others provide a total antioxidant/reducing capacity. Examples of commonly used assays include the ORAC (oxygen radical absorbance capacity) and the TRAP (total radical trapping antioxidant parameter) assays which use fluorescence to test the ability of a compound to neutralize peroxyl radicals. Assays designed to reduce redox metals involve iron, copper and gold reduction. For example, the FRAP (ferric reducing antioxidant power) assay monitors the ability of an antioxidant to reduce Fe(III) to Fe(II) and the CUPRAC (copper reduction antioxidant assay) monitors the ability of an antioxidant to reduce Cu(III) to Cu(II). Each of these assays assess the ability of a compound to interact with one unique ROS (DPPH, ABTS.+, DPPH., superoxide O2.−, H2O2) or a redox metal (Fe, Au, or Cu) and reveal different specificities toward free radicals. Therefore each assay ranks antioxidants in a different order of hierarchy and most interassay comparisons show significant discrepancies, and thus a questionable ranking of the antioxidant power. Most often, the use of multiple, complementary assays is needed to gain a complete understanding of the total antioxidant activity. At present there are no portable antioxidant assays suited for field use. Accordingly, there is a continued demand for simple, easy-to-use tests that can reliably measure the antioxidant capacity of dietary products. Further, there is a continued demand for antioxidant sensing tests that do not require specialized equipment and which can be utilized for high-throughput analysis of a large number of assays.